A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.

The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L. While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis.

The expression of P2Y6 receptor promotes the quality of mucus in colitic mice.

In the intestine, mucins are expressed and secreted by goblet cells and enterocytes in a constitutive manner and in response to secretagogues to form a protective mucus layer. This protective barrier is often lost in inflammatory bowel disease (IBD). Interestingly, extracellular nucleotides, through P2Y receptors, were identified as mucin secretagogues in mucinous epithelia. These nucleotides are found in the intestine's extracellular milieu under basal conditions and in higher concentrations in pathologies such as IBD. It was observed that the mucus layer was affected in P2ry6 knockout mice suffering from dextran sodium sulfate (DSS)-induced colitis. P2ry6-/- mice were more sensitive to DSS-induced colitis, resulting in larger ulcers and increased disease activity index. Interestingly, the absence of P2Y6 receptor expression negatively affected the mucus quality, as shown by a reduction in sulfomucin staining and the absence of a dense internal fucosylated mucin layer in P2ry6-/- mice. Hence, we cannot rule out that the absence of P2Y6 receptors in knockout animals could negatively impact mucin secretion. However, we did not measure a reduction in the number of goblet cells, as previously reported. Instead, the results suggest that goblet cells rapidly discharged mucins to compensate for the mucus layer's increased lability, which resulted in empty goblet cells that are less visible to mucin staining. This study's results, along with previous reports, point toward a protective role for the P2Y6 receptor in IBD.

The P2Y6 receptor signals through Gαq /Ca2+ /PKCα and Gα13 /ROCK pathways to drive the formation of membrane protrusions and dictate cell migration.

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y6 receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y6 during cell migration. Here, we demonstrated that P2Y6 activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y6 increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gαq /calcium/protein kinases C (PKC) and Gα13 /RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y6 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.

The expression of the P2Y6 receptor is regulated at the transcriptional level by p53.

Inflammatory bowel disease (IBD) is a risk factor for the development of colorectal cancer (CRC) for which mutation to p53 is an early event leading to dysplasia. Interestingly, P2RY6 mRNA increases in both pathologies. In this study, we investigated if p53 and p53R273H mutant, commonly found in CRC and IBD, were involved in the transcriptional regulation of P2RY6. First, the P2RY6 promoter was defined as a region corresponding to -1600 to +273 nucleotides relative to the putative TATA-less transcriptional starting site found at position 73,264,505 of NCBI reference sequence NC_000010.11. We cloned this promoter region along with 5'-deletion constructs in the pGL4.10[luc2] vector for luciferase assays to delineate the minimal promoter region. We observed that p53 wt and p53R273H differentially regulated the transcription of the P2RY6 gene. In fact, increasing quantity of p53R273H enhanced the capacity of p53 wt to stimulate the transactivation of the P2RY6 promoter but this cooperative effect was lost when p53R273H was present in a ratio of 3:1. In accordance with the luciferase assays, ChIP analysis revealed that endogenous p53 wt was significantly associated with the P2RY6 proximal promoter, whereas the interaction of the p53R273H with the P2RY6 promoter was not significant. Although further studies are required to fully elucidate the molecular determinant controlling P2Y6 expression in diseases, we propose, for the first time, a molecular mechanism involving a collaboration between p53 wt and p53R273H to regulate the expression of this receptor.

The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis.

Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5'­diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC) was chemically induced in P2ry6 knockout (P2ry6-/-) mice using azoxymethane and dextran sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6-/- mice developed a reduced number of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was significantly lower in P2ry6-/- animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNFα-induced apoptosis. This protective effect was mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP) by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.

The loss of P2X7 receptor expression leads to increase intestinal glucose transit and hepatic steatosis.

In intestinal epithelial cells (IEC), it was reported that the activation of the P2X7 receptor leads to the internalization of the glucose transporter GLUT2, which is accompanied by a reduction of IEC capacity to transport glucose. In this study, we used P2rx7 -/- mice to decipher P2X7 functions in intestinal glucose transport and to evaluate the impacts on metabolism. Immunohistochemistry analyses revealed the presence of GLUT2 at the apical domain of P2rx7 -/- jejunum enterocytes. Positron emission tomography and biodistribution studies demonstrated that glucose was more efficiently delivered to the circulation of knockout animals. These findings correlated with increase blood glucose, insulin, triglycerides and cholesterol levels. In fact, P2rx7 -/- mice had increased serum triglyceride and cholesterol levels and displayed glucose intolerance and resistance to insulin. Finally, P2rx7 -/- mice developed a hepatic steatosis characterized by a reduction of Acaca, Acacb, Fasn and Acox1 mRNA expression, as well as for ACC and FAS protein expression. Our study suggests that P2X7 could play a central role in metabolic diseases.

P2Y2 Receptor Functions in Cancer: A Perspective in the Context of Colorectal Cancer.

Purinergic signaling has recently emerged as a network of signaling molecules, enzymes and receptors that coordinates the action and behavior of cancerous cells. Extracellular adenosine 5' triphosphate activates a plethora of P2 nucleotide receptors that can putatively modulate cancer cell proliferation, survival and dissemination. In this context, the G protein-coupled P2Y2 receptor was identified as one of the entities coordinating the cellular and molecular events that characterize cancerous cells. In this chapter, we will look at the contribution of the P2Y2 receptor in cancer outcomes and use this information to demonstrate that the P2Y2 receptor represents a drug target of interest in the setting of colorectal cancer, for which the role and function of this receptor is poorly defined. More particularly, we will review how the P2Y2 receptor modulates cancer cell proliferation and survival, while promoting cell dissemination and formation of metastases. Finally, we will investigate how the P2Y2 receptor can contribute to the detrimental development of drug resistance that is often observed in cancerous cells.

Multidrug Resistance-Associated Protein 2 Expression Is Upregulated by Adenosine 5'-Triphosphate in Colorectal Cancer Cells and Enhances Their Survival to Chemotherapeutic Drugs.

Extracellular adenosine 5'-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity.

High-throughput analysis of promoter occupancy reveals new targets for Arx, a gene mutated in mental retardation and interneuronopathies.

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations.